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Human cellular nucleic acid-binding protein Zn2+ fingers support replication of human immunodeficiency virus type 1 when they are substituted in the nucleocapsid protein.

ABSTRACT: A family of cellular nucleic acid binding proteins (CNBPs) contains seven Zn(2+) fingers that have many of the structural characteristics found in retroviral nucleocapsid (NC) Zn(2+) fingers. The sequence of the NH(2)-terminal NC Zn(2+) finger of the pNL4-3 clone of human immunodeficiency virus type 1 (HIV-1) was replaced individually with sequences from each of the seven fingers from human CNBP. Six of the mutants were normal with respect to protein composition and processing, full-length genomic RNA content, and infectivity. One of the mutants, containing the fifth CNBP Zn(2+) finger (CNBP-5) packaged reduced levels of genomic RNA and was defective in infectivity. There appear to be defects in reverse transcription in the CNBP-5 infections. Models of Zn(2+) fingers were constructed by using computational methods based on available structural data, and atom-atom interactions were determined by the hydropathic orthogonal dynamic analysis of the protein method. Defects in the CNBP-5 mutant could possibly be explained, in part, by restrictions of a set of required atom-atom interactions in the CNBP-5 Zn(2+) finger compared to mutant and wild-type Zn(2+) fingers in NC that support replication. The present study shows that six of seven of the Zn(2+) fingers from the CNBP protein can be used as substitutes for the Zn(2+) finger in the NH(2)-terminal position of HIV-1 NC. This has obvious implications in antiviral therapeutics and DNA vaccines employing NC Zn(2+) finger mutants.

SUBMITTER: McGrath CF

PROVIDER: S-EPMC165261 | biostudies-literature | 2003 Aug

REPOSITORIES: biostudies-literature

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Human cellular nucleic acid-binding protein Zn2+ fingers support replication of human immunodeficiency virus type 1 when they are substituted in the nucleocapsid protein.

McGrath Connor F CF Buckman James S JS Gagliardi Tracy D TD Bosche William J WJ Coren Lori V LV Gorelick Robert J RJ

Journal of virology 20030801 15

A family of cellular nucleic acid binding proteins (CNBPs) contains seven Zn(2+) fingers that have many of the structural characteristics found in retroviral nucleocapsid (NC) Zn(2+) fingers. The sequence of the NH(2)-terminal NC Zn(2+) finger of the pNL4-3 clone of human immunodeficiency virus type 1 (HIV-1) was replaced individually with sequences from each of the seven fingers from human CNBP. Six of the mutants were normal with respect to protein composition and processing, full-length genom ...[more]

PMID: 12857921

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Project description:The human immunodeficiency virus type 1 (HIV-1) nucleocapsid (NC) protein plays an essential role in several stages of HIV-1 replication. One important function of HIV-1 NC is to act as a nucleic acid chaperone, in which the protein facilitates nucleic acid rearrangements important for reverse transcription and recombination. NC contains only 55 amino acids, with 15 basic residues and two zinc fingers, each having a single aromatic residue (Phe16 and Trp37). Despite its simple structure, HIV-1 NC appears to have optimal chaperone activity, including the ability to strongly aggregate nucleic acids, destabilize nucleic acid secondary structure, and facilitate rapid nucleic acid annealing. Here we combine single molecule DNA stretching experiments with ensemble solution studies of protein-nucleic acid binding affinity, oligonucleotide annealing, and nucleic acid aggregation to measure the characteristics of wild-type (WT) and aromatic residue mutants of HIV-1 NC that are important for nucleic acid chaperone activity. These in vitro results are compared to in vivo HIV-1 replication for viruses containing the same mutations. This work allows us to directly relate HIV-1 NC structure with its function as a nucleic acid chaperone in vitro and in vivo. We show that replacement of either aromatic residue with another aromatic residue results in a protein that strongly resembles WT NC. In contrast, single amino acid substitutions of either Phe16Ala or Trp37Ala significantly slow down NC's DNA interaction kinetics, while retaining some helix-destabilization capability. A double Phe16Ala/Trp37Ala substitution further reduces the latter activity. Surprisingly, the ensemble nucleic acid binding, annealing, and aggregation properties are not significantly altered for any mutant except the double aromatic substitution with Ala. Thus, elimination of a single aromatic residue from either zinc finger strongly reduces NC's chaperone activity as determined by single molecule DNA stretching experiments without significantly altering its ensemble-averaged biochemical properties. Importantly, the substitution of aromatic residues with Ala progressively decreases NC's nucleic acid chaperone activity while also progressively inhibiting viral replication. Taken together, these data support the critical role of HIV-1 NC's aromatic residues, and establish a direct and statistically significant correlation between nucleic acid chaperone activity and viral replication.

Dataset Information

Human cellular nucleic acid-binding protein Zn2+ fingers support replication of human immunodeficiency virus type 1 when they are substituted in the nucleocapsid protein.

Publications

Human cellular nucleic acid-binding protein Zn2+ fingers support replication of human immunodeficiency virus type 1 when they are substituted in the nucleocapsid protein.

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